CENP-A is a centromere-specific histone H3 variant. CENP-A is required for specifying centromeric loci, where it is thought to nucleate assembly of the kinetochore, the protein complex that mediates chromosome segregation during mitosis. Failures in chromosome segregation result in at least 45% of spontaneous abortions, many kinds of cancer, and diseases such as Down's syndrome (Jacobs and Hassoid, 1995; Mitelman, 1994). It is not known how CENP-A is targeted specifically to centromeric loci. One hypothesis of centromere specification is that CENP-A is epigenetically inherited. This hypothesis will be tested by assembling purified recombinant Xenopus CENP-A (XCENP-A) in Xenopus egg extracts, to facilitate assays for the stability of XCENP-A chromatin during DNA replication in vitro. If this hypothesis is correct, the presence of XCENP-A is necessary to ensure XCENP-A assembly in future rounds of DNA replication on any substrate. An alternative hypothesis is that XCENP-A assembly is restricted to centromeric loci by post-translational modifications and/or modification of the DNA itself. The candidate has prior experience studying histone post-translational modifications. Reagents are already available in the Newport lab to test a potential requirement for DNA methylation in restricting XCENP-A assembly. Results of the studies proposed here will be critically important for future studies on kinetochore assembly and creation of artificial chromosomes in vitro. These studies will also have direct implications for understanding cancer causation, and applications in gene therapy.